Process of quality examining for microarray of biological material

ABSTRACT

The present invention relates a process of quality examining for microarray of biological material. More particularly, the present invention is directed to a process of quality examining for microarray of biological material, which comprises 1) a step for mixing probe and a compound which emits light or heat and does not react with said probe, 2) a step for microarraying the mixture obtained in step 1) on a substrate, and 3) a step for measuring light or heat emitted by the scanning of each spots of the microarray mixture.

TECHNICAL FIELD

[0001] The present invention relates a process of quality examining formicroarray of biological material. More particularly, the presentinvention is directed to a process of quality examining for microarrayof biological material, which comprises a step for mixing probe and acompound which emits light or heat and does not react with said probe, astep for microarraying the mixture obtained in step 1) on a substrate,and a step for measuring light or heat emitted by the scanning of eachspots of the microarray mixture.

BACKGROUND ART

[0002] A microarray of biological material means a device for detectinggenes or proteins wherein a large number of fragments of DNA, RNA orprotein, are immobilized on a small substrate in high density. Suchmicroarray chip is applied to researches for analyzing DNA mutations,RNA expressions and functions of proteins for understanding function ofgenes in large scale.

[0003] The microarray of a biological material (hereinafter, Biochip)may be classified into cDNA chip, oligonucleotide chip and protein chipon the basis of size of biological materials immobilized thereon.Full-length open leading frames or expressed sequence tag(EST)s havingmore than five (5) hundred base pairs can be immobilized on the cDNAchip. Oligonucleotides consisting of approximately 15 to 25 base pairscan be immobilized on the oligonucleotide chip.

[0004] The biochip on which plasmid DNA probe, oligonucleotide probe orprotein is immobilized is primarily used for detecting gene expression,protein activity and DNA mutation. The most general applications of DNAchip, are for observing the difference of RNA expression between normalcell and abnormal cell. For example, researches for analyzing thedifference of RNA expression between human normal cell and cancer cellare performed. Various studies employing high-density oligonucleotidechips, have been performed to analyze the type and system of geneexpression of Saccharomyces cerevisea of which total six (6) thousandgenes had been fully discovered.

[0005] Meanwhile, probes microarrayed on the biochip, are plasmid DNAcomprising cDNA, products of polymerase chain reaction or syntheticoligonucleotide.

[0006] Probes can be microarrayed on the substrate throughphotolithography (Affymetrix Inc.), non-contact printing method usingInk-Jet injection process (Piezoelectric printing; Packard InstrumentInc., Syringe-solenoid printing; Cartesian Techonologies), contactprinting method (Quill and Splite Pin; Telechem International Inc., Pinand Ring; Genetic Microsystem, Capillary pin; Bioneer Corp.) and so on.

[0007] However, whatever microarraying method is used, the amount ofprobes arrayed in each spot or in each biochip cannot be uniform andalso the feature of spot microarrays can not be regular due to theeffects caused from types of slide glass, electric noise generated inmeasuring wavelength and evaporation of spot.

[0008] More specifically, the direct synthesis of oligo-type probes onthe substrate through photolithography process has critical problem thatoligomers thus prepared on the substrate directly are not homogeneouseven in same spot due to the failure of synthetic reaction.

[0009] Further, the fluctuation of the amount and feature ofmicroarrayed probes may cause critical error especially in interpretingresults of observing the difference of RNA expression pattern. Theuniformity and regularity of microarrayed probes also should be securedin case of a biochip used for detecting DNA mutation accurately.

[0010] Furthermore, the conrol of the above uniformity and regularityare very important for the research on that DNA mutation or RNAexpression should be observed continuously through same probes by usingseveral biochips.

[0011] Therefore, the step for controlling the uniformity and regularityof the amount of probes should be necessarily added to a process formanufacturing biochip. In the addition, it is needed to provide thecorrection factor that indicate such differences between microarrayedfor the bio-chip users in order to secure high fidelity of a bio-chiptest result through the correction of the hybridization results.

[0012] Therefore, the primary object of the present invention is toprovide a process of quality examining for microarray of biologicalmaterial, which comprises a step for mixing probe and a compound whichemits light or heat and does not react with said probe, a step formicroarraying the mixture obtained in step 1) on a substrate, and a stepfor measuring light or heat emitted by the scanning of each spots of themicroarray mixture.

[0013] The other object of the present invention is to provide amicroarray of biological material wherein a mixture of probes and acompound which emits light or heat and does not react with said probe,is micrroarryed on substrate.

DISCLOSURE OF THE INVENTION

[0014] The object of the present invention is achieved by providing aprocess of quality examining for microarray of biological material,which comprises:

[0015] 1) a step for mixing probe and a compound which emits light orheat and does not react with said probe;

[0016] 2) a step for microarraying the mixture obtained in step 1) on asubstrate; and

[0017] 3) a step for measuring light or heat emitted by the scanning ofeach spots of the microarray mixture.

[0018] In addition, the above method of the present invention mayfurther comprise a step 4) for calibrating the intensity of light orheat emitted from the hybridized microarray of biological material byusing the intensity of light or heat measured in said step 3).

[0019] Bio-chip users of the present invention can further perform astep for measuring the intensity of light or heat emitted fromhybridization of microarrayed probe and target and a step forcalculating a correction factor by using the above-measured intensity oflight or heat to exclude error caused by discrepancy between size ofeach spots, in order to get more accurate experimental results.

[0020] The other object of the present invention is to provide amicroarray of biological material wherein a mixture of probes and acompound which emits light or heat and does not react with said probe ismicrroarryed on substrate.

[0021] Probes of the present invention are selected from the groupconsisting of biological materials formed by linear or circularcombinations of peptides including amino acid, nucleic acid,polysaccharide and phospholipid, respectively.

[0022] In addition, A compound of the present invention which emitslight or heat and does not react with the probe is preferably selectedfrom the group consisting of fluorescent material, chemi-luminescentmaterial, bio-luminescent material, calorimetric material andlight-scattering material, and is preferably selected from the groupconsisting of rhodamine derivatives and fluoresceine derivativesincluding Fluorescein, Coumarin,4′,5′-Dichloro-2′,7′-dimethoxyfluorescein, Tetramethylrhodamine,X-rhodamine, Eosin, Oregon Green, Rhodamine Green, Rhodamine Red, TexasRed and Rodamine and so on, especially in the case that the biologicalmaterial is nucleic acid.

[0023] Two types of rhodamine (6-carboxyrhodamine 6G; 6-CR6G,5-carboxyrhodamine 6G; 5-CR6G) of the present invention having followingstructures is activated in 520 nm and emit fluorescence of which wavelength is 545 nm.

[0024] In addition, the rhodamines do not react with DNA, RNA andprotein, and may be washed easily by water or other aqueous solutionafter measuring the uniformity of microarray of biological material.Therefore, the rhodamines have the advantages that general chip scannermay be used without any extra equipment and subsequent processsincluding hybridization is not affected thereby.

[0025] <Structural Formulas>

[0026]FIG. 1 show a process of quality examining for microarray ofbiological material by using rhodamine 6G.

[0027] More particularly, the predetermined amount of rhodamine is mixedwith the predetermined amount of probe, and the mixture is microarrayed.The microarray of the mixture is examined the uniformity of each spotthrough measuring the intensity of fluorescence, and the result ofexamination is expressed numerically.

[0028] Finally, bio-chip users are provided the numerical data alongwith a biological chip in order to interpret the result more accurately,in DNA chip research.

BRIEF DESCRIPTION OF THE DRAWINGS

[0029] The above objects and other advangages of the present inventionwill become more apparent by describing in detail a preferred embodimentthereof with reference to the attached drawings, in which:

[0030]FIG. 1. shows a process which the results of hybridization arenormalized in order to secure high quality of a biological chip.

[0031]FIG. 2. shows microarrays wherein oligonucleotides are mixed withrhodamine diluted in the predetermined ratio.

[0032]FIG. 3. shows microarrays wherein thirty oligonucleotides (30 mer)are mixed with the same amount of rhodamine.

[0033]FIG. 4. shows what effect a process for checking the regularity ofthe amount of probes has on the results of hybridization.

[0034]FIG. 5. shows microarrays which are made after mixtures ofoligonucleotide and rhodamine are diluted twice by two fold.

[0035]FIG. 6. shows a regressive equation which indicates relationshipbetween fluorescence of microarrays after microarraying and fluorescenceof microarrays after hybridization.

BEST MODE FOR CARRYING OUT THE INVENTION

[0036] Hereinafter, the present invention will be described in greaterdetail with reference to the following examples. The examples are givenfor illustration of the invention and not intended to be limiting thepresent invention.

EXAMPLE 1

[0037] Determining the Proper Concentration of Rhodamine 6G for thePurpose of Quality Examining for Microarray of Biological Material

[0038] A rhodamine used for obtaining the data to exclude error causedby discrepancy between size of each spot, should meet the followingconditions.

[0039] First, the concentration of rhodamine should be in the rangewhich can be detected by scanner. That is, the concentration ofrhodamine should not too low to be detected and too high not to besaturated.

[0040] Second, the rhodamine of the present invention should have theconcentration in which rhodamine can be washed easily, with meeting theabove condition.

[0041] Third, the rhodamine of the present invention should have theconcentration in which subsequent process after washing includinghybridazation is not affected.

[0042] Therefore, the following embodiment was performed in order todetermined the most appropriate concentration of rhodamine 6G to meetthe above three (3) conditions.

[0043] A. Preparation of Rhodamine 6G

[0044] 10 mM rhodamine 6G was dissolved in methylalchol, and 10 μl ofthe solution was diluted in 990 μl Sodium Borate buffer (pH9.0; SBbuffer) so that the concentration could be 100 pmol/μl.

[0045] 100 pmol/μl solution was diluted in SB buffer sequentially, andsolutions having eleven (11) kinds of concentration are prepared (seeFIG. 2).

[0046] B. Microarray of the Mixture of Rhodamine and Oligonucleotide

[0047] Each diluted solution was mixed with 10 pmol/μl oligonucleotide.Each mixture was microarrayed on a acrylamide gel pad by usingHT-Arrayer™ (Bioneer Corp, Korea) by fourteen times (14), respectively.The gel pad was made through smearing 30 μl of the solution composed of0.1% glycidyl metacrylate, 8% acrylamide, 1/20 ammonium persulfate and1/100 N,N,N′,N′-tetramethylethylenediamine(TEMED) on a washed slideglass.

[0048] C. Scanning and Washing

[0049] Rhodamine contained in microarrayed spots was activated in 532 nmby using a chip scanner (GenePix4000, Axon Instrument Inc, USA), and wasscanned in PMT 600(Photomultipler tubes 600) in order to obtain thefluorescence data.

[0050] After that, the chip was washed by water in room temperature forfive (5) minutes, and re-scanned in same PMT 600 in same wavelength. Theresults of scanning were illustrated in FIG. 2.

[0051] As illustrated in FIG. 2, 10 pmol/μl oligonuceotide was mixedwith rhodamine 6G which was diluted by the predetermined ratio ofdilution, and the mixture was microarrayed.

[0052] a was the result of scanning rhodamine 6G activated in 532 nm,and b was the result of scanning in different conditions, for examplethe condition that a microarray was washed by water in room temperaturefor five (5) minutes, when increasing the amount of rhodamine which wasmixed with oligonucleotide composed thirty (30) monomers.

[0053] Rhodamine of which concentration was 1 fmol/μl in lane 1, 5fmol/μl in lane 2, 10 fmol/μl in lane 3, 50 fmol/μl in lane 4, 100fmol/μl in lane 5, 500 fmol/μl in lane 6, 1 pmol/μl in lane 7, 5 pmol/μlin lane 8, 10 pmol/μl in lane 9, 50 pmol/μl in lane 10 and 100 pmol/μlin lane 11, was mixed with 10 pmol/μl of oligonucleotide composed ofthirty (30) monomers respectively, and each mixture was microarrayedrepeatedly fourteen (14) times. The mean diameter of a spot was 180 μmand the mean distance of center to center was 150 μm.

[0054] As described in FIG. 2, the intensity of fluorescence wassaturated in the case that the concentration of rhodamine was not lessthan 500 fmol/μl. In addition, the intensity of fluorescence was stilldetected after washing in the case that the concentration of rhodaminewas not less than 1 pmol/μl, because rhodamine was not eliminatedthrough simple washing.

[0055] Based on the above results, 100 fmol/μl was selected as theappropriate concentration that the intensity of fluorescence was notsaturated and rhodamine was sufficiently eliminated through simplewashing for five (5) minutes.

[0056] D. Selection of the Washing Condition

[0057] It was examined whether 100 fmol/μl rhodamine selected above waseliminated easily by blocking solution (10% ethanolamine) appliedgenerally to a gel pad chip or not.

[0058] As illustrated in FIG. 3, 100 fmol/μl rhodamine was mixed with 10pmol/μl oligonucleotide composed of thirty (30) monomers, and themixture was microarrayed.

[0059] a was the results of scanning spots in 532 nm without anytreatment after microarraying. b was the results of scanning spots inPMT 600(photomultiplier tubes 600) and c was the results of scanningspots in PMT 900(photomultiplier tubes 900), after washing a microarrayby 10% ethanolamine for five (5) minutes.

[0060] As results, 100 fmol/μl rhodamine was completely eliminated whenwashing the microarry by 10% ethanolamine in room temperature for five(5) minutes. That is, no intensity of fluorescence was detected in PMT600 and in PMT 900. The above results show the advantages of the presentinvention that an experiment takes less time because a microarry can bewashed easily without the addition of extra washing process.

[0061] According to the above results, 100 fmol/μl was selected as themost appropriate concentration and washing by water or 10% ethanolaminein room temperature for five (5) minutes was selected as the washingcondition.

EXAMPLE 2

[0062] The effect of Rhodamine 6G on the Step of Hybridization

[0063] Hybridization was performed in order to confirm whether the mostappropriate concentration and the washing condition selected in Example1 were selected properly.

[0064] A. Preparation of the Mixture of Rodamine and Oligonucleotide

[0065] 10 pmol/μl of two types of oligonucleotide was mixed with 100fmol/μl rhodamine respectively, and the two types of the mixture wasmixed with each other sequentially in the ratio from 1:0 to 1.5:8.5(FIG.4).

[0066] B. Microarray of the Mixture of Rhodamine and Oligonucleotide

[0067] Each Mixture was microarrayed on a acrylamide gel pad by usingHT-Arrayer™ (Bioneer Corp, Korea) by nineteen (19) times. The gel padwas made through smearing 30 μl of the solution composed of 0.1%glycidyl metacrylate, 8% acrylamide, 1/20 ammonium persulfate and 1/100N,N,N′,N′-tetramethylethylenediamine(TEMED) on a slide glass.

[0068] C. Scanning and Washing

[0069] Rhodamine contained in microarrayed spots was activated in 532 nmby using a chip scanner (GenePix4000, Axon Instrument Inc, USA), and wasscanned in PMT 600(Photomultipler tubes 600) in order to obtain thefluorescence data. After that, the chip was washed by 10% ethanolaminein room temperature for five (5) minutes.

[0070] D. Hybridization

[0071] Two types of microarrayed oligonucleotide were hybridized witheach target that was completely complementary to each oligonucleotideand of which 5′-terminal was labeled by cy3 or cy5. The target labeledby 1 pmol fluorescent material was mixed with 20 μl hybridizing buffer(1M NaCl, 1 mM EDTA, 1% Tween 20 and 5 mM sodium phosphate), and reactedwith each oligonucleotide in 40° C. for 1 hour. non-reacted targets andnon-specific products of hybridization were eliminated through washingby hybridizing buffer diluted by ten (10) fold in 65° C. for 15 minutes.

[0072] E. Scanning

[0073] The intensity of fluorescence of hybridization-product wasmeasured by using a chip scanner (GenePix4000, Axon Instrument Inc,USA), after Cy3 was activated in 532 nm and cy5 was activated in 650 nm.

[0074] 10 pmol/μl of two types of oligonucleotide (comp-cy3 andcomp-cy5) was mixed with each other, and the mixture was mixed with 100pmol/μl rhodamine, and the final mixture was microarrayed (FIG. 4a).

[0075] In addition, the microarray was activated in 532 nm, and waswashed by ethanolamine in room temperature for five (5) minutes. Afterwashing, the above two (2) kinds of oligonucleotide were hybridized witheach target oligonucleotide that was completely complementary to eachprobe and was labeled by cy3 or cy5.

[0076] b was the result of measuring the intensity of fluorescence aftercy3 was activated in 532 nm and cy5 was activated in 635 nm. Green colorindicates fluorescence resulted from cy3, and red color indicatesfluorescence resulted from cy5, and yellow color indicates the mixtureof the above two fluorescence. 10 pmol/μl comp-cy3 and 10 pmol/μlcomp-cy5 was mixed with each other sequentially in the ratio from 1:0 to1.5:8.5, and the mixture was microarrayed in lane 1 to lane 18 of FIG. 4after mixed with 10 pmol/μl rhodamine.

[0077] As results, the feature of yellow color indicating the mixture oftwo colors by fluorescence corresponded with the mixing ratio of twoprobe-oligonucleotides.

[0078] This results showed that rhodamine using for quality examining ofmicroarray of biological material had no effect on a step forhybridization, especially on a step for competitive hybridization whichtwo targets labeled with two kinds of fluorescent material respectivelywere hybridized in one spot.

[0079] That is, the above results show that rhodamine 6G can be used asthe material for quality examining for the microarray of biologicalmaterial because rhodamine does not cause any error in a process foranalyzing genes.

EXAMPLE 3

[0080] Exclusion of Error Caused by Discrepancy Between Size of EachSpots

[0081] This embodiment was performed in order to show a simple exampleof calculating a correction factor which can exclude error caused bydiscrepancy between size of each spots. However, the method forcalculating a correction factor is not limited to this embodiment.

[0082] A. Preparation of the Mixture of Rodamine and Oligonucleotide

[0083] 100 pmol/μl of one (1) kind of oligonucleotide was mixed with thesame amount of 100 fmol/μl rhodamine 6G. The mixture was diluted in SBbuffer by two-fold and by four-fold, and each diluted solution wasmicroarrayed three times (FIG. 5a).

[0084] The non-diluted solution was microarrayed in lane 1, lane 2 andlane 3 of FIG. 5. The two-fold diluted solution was microarrayed in lane4, lane 5 and lane 6, and the four-fold diluted solution wasmicroarrayed in lane 7, lane 8 and lane 9.

[0085] B. Scanning and Washing

[0086] Rhodamine contained in microarrayed spots was activated in 532 nmby using a chip scanner (GenePix4000, Axon Instrument Inc, USA), and wasscanned in PMT 600(Photomultipler tubes 600) in order to obtain thefluorescence data. After that, the chip was washed by 10% ethanolaminein room temperature for five (5) minutes.

[0087] C. Hybridization

[0088] The microarrayed oligonucleotide was hybridized with the targetthat was completely complementary to the oligonucleotide and of which5′-terminal was labeled by cy3. The target labeled by 1 pmol fluorescentmaterial was mixed with 20 μl hybridizing buffer (1M NaCl, 1 mM EDTA, 1%Tween 20 and 5 mM sodium phosphate), and reacted with theoligonucleotide in 40° C. for 1 hour. Non-reacted targets andnon-specific products of hybridization were eliminated through washingby hybridizing buffer diluted by ten (10) fold in 65° C. for 15 minutes.

[0089] D. Scanning

[0090] The intensity of fluorescence of hybridization-product wasmeasured by using a chip scanner (GenePix4000, Axon Instrument Inc,USA), after Cy3 was activated in 532 nm (FIG. 5b). Table 1 shows themean intensity of fluorescence obtained respectively after microarrayingand after hybridization. Each spot had the area of 7850 μm², and thesemi-diameter of 50 μm.

[0091] The intensity of fluorescence of rhodamine obtained aftermicroarraying was linearly proportioned to the concentration ofmicroarrayed oligonucleotide (y=541,63x−12861, R²=0.9453). In addition,the intensity of fluorescence of rhodamine obtained after microarrayinghighly correlated with the intensity of fluorescence of cy3 obtainedafter hybridization (R=0.9096).

[0092] That is, this results shows that the discrepancy between theamount of microarrayed probes has direct effect on the finalhybridization result. Therefore, the final users of a biological chipshould be provided the date which can exclude error caused by the abovediscrepancy in order to correct the final hybridization result. Table 1shows the mean intensity of fluorescence respectively obtained aftermicroarraying and after hybridization for spots in FIG. 5.

[0093]FIG. 6 shows the results of calibrating the regressive equationwith respect to the relationship between the intensity of thefluorescence obtained after microarraying and the intensity of thefluorescence obtained after hybridization, by using the mean intensityof fluorescence emitted in the area of 7850 μm² after the intensity offluorescence in each spot was obtained in 532 nm.

[0094] The regressive equation was Y(the intensity of fluorescenceobtained after hybridization)=2836.6Ln(X;the intensity of fluorescenceobtained after microarraying)−18970(R=0.9646).

[0095] The intensity of fluorescence of cy3 was inferred from theintensity of fluorescence of rhodamine 6G of which range was 1,000 to100,000 by using the above regressive equation. The discrepancy betweenthe intensity of fluorescence of rhodamine 6G means the discrepancybetween the amount of microarrayed probes, for example oligonucleotide.The intensity of fluorescence of rhodamine 6G of which range was 1,000to 100,000 can cover almost possible variation thereof.

[0096] The arithmetic mean of the intensity values of fluorescenceobtained after microarraying was calculated and was converted to theintensity of fluorescence obtained after hybridization (11749.61).

[0097] The intensity values of fluorescence obtained after hybridizationwas subtract from the value calculated by conversion (11749.61). Thevalues calculated by subtraction were used as correction factors, andthe values calculated by adding the correction factor to the actualintensity of fluorescence are always 11749.61.

[0098] More particularly, the variation in the amount of probes amongspots caused the variation in the intensity of fluorescence ofrhodamine, in the case that probes having the predeterminedconcentration are mixed with 100 fmol/μl rhodamine and the mixture ismicroarrayed. Finally, the variation in the intensity of fluorescence ofrhodamine has the effect on the intensity of fluorescence obtained afterhybridization.

[0099] For example, assuming that the intensity of fluorescence emittedin spot A is 1000 after microarraying and 1000 after hybridization andthe intensity of fluorescence emitted in spot B is 2000 aftermicroarraying and 1500 after hybridization, it can be misunderstood thatthe amount of hybridized target in spot B is larger than the amount ofhybridized target in spot A when judging only by the actual intensity offluorescence after hybridization.

[0100] However, such discrepancy between the intensity of fluorescencecan be corrected by subtracting the discrepancy resulting from theamount of microarrayed probes in order to compare the intensity offluorescence accurately.

[0101] In the case that each appropriate correction factor, 1125.07 and9158.89 is added to each intensity of fluorescence in spot A and in spotB, the intensity of fluorescence is 1225.07 in spot A and 11158.89 inspot B. Therefore, assuming that the same amount of probes is containedin spot A and in spot B, it could be concluded that the amount ofhybridized target in spot A is larger than the amount of hybridizedtarget in spot B.

[0102] As explained the above, the correction factor can be necessarilyused in order to interpret the final results more accurately. Table 2shows the correction factors calculated by the regressive equation(Y(the intensity of fluorescence obtained afterhybridization)=2836.6Ln(X; the intensity of fluorescence obtained aftermicroarraying)−18970). TABLE 1 The mean intensity of fluorescenceaccording to each concentration of oligonucleotide after hybridizationthe concentration of the mean intensity of the mean intensitymicroarrayed fluorescence obtained of fluorescence oligonucleotide in532 nm after obtained in 532 nm No. (pmole/ul) microarraying afterhybridization 1 100 50308 11889 2 100 38592 10630 3 100 38254 10848 4 5012190 8069 5 50 9338 6322 6 50 11395 7952 7 25 3576 5497 8 25 2534 30909 25 2423 2232

[0103] TABLE 2 The correction factors calculated by the regressiveequation (Y = 2836.6 Ln(X)- 18970) the mean the mean intensity ofintensity of the mean normalized fluorescence fluorescence intensity ofobtained in obtained in fluorescence obtained 532 nm after 532 nm aftercorrection in 532 nm after microarraying hybridization factorhybridization 1000 624.5386244 1125.07138 11749.61 2000 2590.7199179158.890083 11749.61 3000 3740.862242 8008.747758 11749.61 40004556.901209 7192.708791 11749.61 5000 5189.870207 6559.789798 11749.616000 5707.048535 6042.566465 11749.61 7000 6144.307353 5605.30264711749.61 8000 6525.082501 5226.527499 11749.61 9000 6857.185864892.42414 11749.61 10000 7156.051499 4593.558501 11749.61 110007426.408355 4323.201645 11749.61 12000 7673.224827 4076.385173 11749.6113000 7900.273972 3849.336028 11749.61 14000 8110.488840 3639.12135411749.61 15000 8306.193825 3443.416175 11749.61 16000 8489.2637943280.346206 11749.61 17000 8861.231596 3088.373404 11749.61 180008823.367153 2926.242847 11749.61 19000 8976.724233 2772.875767 11749.6120000 9122.232792 2627.77208 11749.61 21000 9260.630971 2488.97902911749.61 22000 9392.580648 2357.020352 11749.61 23000 9518.6615172230.928483 11749.61 24000 9639.40612 2110.20388 11749.61 250009755.201789 1994.208211 11749.61 26000 9866.456264 1883.154736 11749.6127000 9973.509478 1776.100522 11749.61 28000 10076.66994 1672.94006211749.61 29000 10176.20998 1573.400024 11749.61 30000 10272.375121477.234883 11749.61 31000 10365.38873 1384.223271 11749.61 3200010455.44509 1294.164914 11749.61 33000 10549.73197 1206.878027 11749.6134000 10627.41289 1122.197112 11749.61 35000 10709.63894 1039.97106411749.61 36000 10789.54845 960.0615548 11749.61 37000 10867.26838882.3416246 11749.61 38000 10942.91553 806.694475 11749.61 3900011016.59759 733.0124102 11749.61 40000 1108.41408 661.1959161 11749.6141000 11158.45715 591.1528512 11749.61 42000 11226.81226 523.797786411749.61 43000 11293.55887 456.0511275 11749.61 44000 11358.77094360.8390601 11749.61 45000 11422.51744 327.0925572 11749.61 4600011484.86281 264.7471904 11749.61 47000 11545.86731 203.7426886 11749.6148000 11605.58741 144.0225881 11749.61 49000 11664.07608 85.5339180211749.61 50000 11721.38308 28.22691844 11749.61 51000 11777.55521−37.84521415 11749.61 52000 11832.63656 −137.0586663 11749.61 5300011886.66867 −137.0586663 11749.61 54000 11939.69077 −190.080770811749.61 55000 11991.73994 −242.1299376 11749.61 56000 12042.85123−293.2412303 11749.61 57000 12093.05785 −343.4478507 11749.61 5800012142.39127 −392.7812681 11749.61 59000 12190.88134 −441.271387711749.61 60000 12238.55641 −488.9464096 11749.61 61000 12285.44343−535.8834275 11749.61 62000 12331.58802 −581.958021 11749.61 6300012376.95459 −827.3445892 11749.61 64000 12421.62638 −672.016378611749.61 65000 12465.60555 −715.9965541 11749.61 66000 12508.91327−759.3032856 11749.61 67000 12551.56971 −801.9597085 11749.61 6800012593.59418 −843.9841809 11749.61 69000 12635.00514 −885.395135311749.61 70000 12675.82023 −926.210228 11749.61 71000 12716.05636−953.4463636 11749.61 72000 12755.72974 −1006.119738 11749.61 7300012794.85588 −1045.245875 11749.61 74000 12833.44967 −1063.83966811749.61 75000 12871.52543 −1121.915407 11749.61 76000 12909.09682−1159.486817 11749.61 77000 12946.17708 −1196.567084 11749.61 7800012982.77888 −1233.168882 11749.61 79000 13018.9144 −1269.304403 11749.6180000 13054.59538 −1304.985378 11749.61 81000 13089.8331 −1340.32309711749.61 82000 13124.63844 −1375.028441 11749.61 83000 13159.02189−1409.411892 11749.61 84000 13192.99356 −1443.383556 11749.61 8500013226.56318 −1476.953179 11749.61 86000 13259.74016 −1510.13016511749.61 87000 13292.53359 −1542.923594 11749.61 88000 13324.95223−1575.342232 11749.61 89000 13357.00455 −1607.394551 11749.61 9000013388.69874 −1639.088735 11749.61 91000 13420.0427 −1670.432701 11749.6192000 13451.0441 −1701.434102 11749.61 93000 13481.71035 −1732.10034711749.61 94000 13512.0486 −1762.438604 11749.61 95000 13542.06582−1792.455815 11749.61 96000 13571.7687 −1822.158704 11749.61 9700013601.16370 −1851.553786 11749.61 98000 13630.25737 −1880.64737411749.61 99000 13659.06559 −1909.445591 11749.61 100000 13687.56437−1937.954374 11749.61

INDUSTRIAL APPLICABILITY

[0104] As explained the above, final users can be provided ahigh-quality biological chip through introducing a process of qualityexamining for microarray of a biological material of the presentinvention to mass production process of a biological chip. In addition,bio-chip users of the present invention can further perform a step formeasuring the intensity of light or heat emitted from hybridizedmicroarray of biological material and a step for calculating acorrection factor by using the above-measured intensity of light or heatto exclude error caused by discrepancy between size of each spots, inorder to get more accurate experimental results.

What is claimed is:
 1. A process of quality examining for microarray of biological material, which comprises: 1) a step for mixing probe and a compound which emits light or heat and does not react with said probe; 2) a step for microarraying the mixture obtained in step 1) on a substrate; and 3) a step for measuring light or heat emitted by the scanning of each spots of the microarrayed mixture.
 2. A process of quality examining for microarray of biological material according to claim 1, which further comprises a step for calibrating the intensity of light or heat emitted from the hybridization of microarray of biological material by using the intensity of light or heat measured in said step 3).
 3. A process of quality examining for microarray of biological material according to claim 2, which further comprises a step for measuring the intensity of light or heat emitted from hybridized microarray of biological material, and a step for calculating a correction factor by using the above-measured intensity of light or heat to exclude error caused by discrepancy between size of each spots.
 4. A process of quality examining for microarray of biological material according to claim 1, characterized in that said probe is selected from the group consisting of biological materials formed by linear or circular combinations of peptides including amino acid, nucleic acid, polysaccharide and phospholipid, respectively.
 5. A process of quality examining for microarray of biological material according to claim 1, wherein the light or heat-emitting compound which does not react with the probe is selected from the group consisting of fluorescent material, chemi-luminescent material, bio-luminescent material, calorimetric material and light-scattering material.
 6. A process of quality examining for microarray of biological material according to claim 5, wherein said biological material is nucleic acid and the light or heat-emitting compound which does not react with the probe is selected from the group consisting of Fluorescein, Coumarin, 4′,5′-Dichloro-2′,7′-dimethoxyfluorescein, Tetramethylrhodamine, X-rhodamine, Eosin, Oregon Green, Rhodamine Green, Rhodamine Red, Texas Red and Rodamine.
 7. A microarray of biological material wherein a mixture of a probe and a compound which emits light or heat and does not react with said probe is micrroarryed on substrate. 